The sphingomyelin (SM) pathway is a highly conserved, ubiquitous cell-signaling pathway. Ceramide, the central molecule in this pathway, serves as a second messenger in the regulation of a variety of cellular functions including proliferation, differentiation, cell cycle arrest and apoptosis. Our long-term goal is to identify critical sub-cellular targets of ceramide in radiation-induced apoptosis and alter radiation sensitivity. The objective of this proposal is to identify critical changes in lipids targeted to the mitochondria and in mitochondrial lipids themselves, in response to ionizing radiation-induced cell death. The central hypothesis to be tested is that increased mitochondrial ceramide induces mitochondrial permeability changes during ionizing radiation induced apoptosis. The rationale behind the proposed research is that changes in mitochondrial lipid levels that are critical to this organelle, (in addition to pro- and anti-apoptotic protein components), likely participate in the membrane driven physiological events that occur during apoptosis. Therefore, the mitochondrial lipid environment must be investigated before changes in permeability; the release of cytochrome c and/or the generation of ROS from this organelle can be fully appreciated and understood. [unreadable] To accomplish the objective of this application, we will pursue three Specific Aims: (1) Test the hypothesis that ceramide has a critical role in ionizing irradiation induced mitochondrial membrane permeability. (2) Test the hypothesis that specific biochemical steps in the apoptotic pathway are critical in inducing mitochondrial ceramide increases. (3) Test the hypothesis that ceramide acts to induce cell killing by free radicals. These results should provide valuable information for the identification of novel molecular targets for enhancing or decreasing ionizing radiation-induced apoptosis. [unreadable] [unreadable]